Ligands of the translocator protein, TSPO, are sensitive and specific biomarkers and powerful imaging agents of neurological damage. But in spite of more than 30 years of study, the function of this ancient conserved protein that is ubiquitously expressed in all kingdoms of life, remains unclear. A common theme in many different organisms is upregulation of TSPO in response to stress. In this context, we are proposing a new role for TSPO as a promoter of neuroprotection via activation of M2 microglia cells, which play a major role in neuroinflammatory diseases such as Alzheimer?s and Parkinson?s. Our goal is to characterize the activation and stress responses of microglia cells from human and mouse and to explore the role of TSPO and its ligands in this response. We will use RNAseq to determine a common expression signature and CRISPER Cas9 to test the effects of TSPO deletion and mutation on that signature (Aim1). An important tool for guiding and empowering this effort is the availability of high resolution crystal structures of the bacterial homolog of human TSPO from Rhodobacter sphaeroides, whose structure we have solved in a lipidic environment in native and mutant forms. This protein is well conserved and accessible for mutagenesis, purification, crystallization and biochemical analysis. Further, a functional assay has been developed based on Rhodobacter?s TSPO-dependent resistance to metal toxicity, which will allow us to screen mutant forms as well as various ligands to guide our intervention in the microglial system (Aim 2). Although first recognized in mitochondria as an outer membrane protein that bound benzodiazepine drugs, mounting evidence suggests TSPO involvement in a number of complex cellular processes, including cholesterol transport, porphyrin transport, tumor progression, neuroinflammation and Alzheimer?s disease. The strength of the proposal is the structure-guided comparative analysis of several systems in which TSPO is highly induced in association with inflammation and stress. This common theme among different organisms holds the potential for revealing unique insights into mechanism. A clear mechanistic relationship between TSPO structure and function will provide the basis for developing novel structure-based ligands for detection and treatment of neurological disease.